Fig. 3

NiPT inhibits proteasome function in CML cells. a NiPT accumulates proteasome substrate proteins in CML cells. Cells were treated with various doses of NiPT for 6 h. The protein levels of PARP, ubiquitinated proteins (Ubs), and p27 were detected using western blots. Actin was used as a loading control. b NiPT has no obvious effect on the 20S proteasome peptidase activities in KBM5 and KBM5R cells. Cell lysate was treated with NiPT, and then the CT-like activity at different times was recorded using the fluorogenic Suc-LLVY-AMC substrate. Mean ± SD (n = 3). c Active-site-directed labeling of proteasomal DUBs. Cells were treated with NiPT, and then the cell lysate-contained DUBs were labeled with HA-UbVS. Labeled HA was detected via western blotting. b-AP15 (0.15 μM) was used as a positive control. d NiPT inhibits the phosphorylation of USP14. Cells were treated with the indicated dose of NiPT for the indicated duration. p-USP14 and total USP14 were detected with western blot. e NiPT treatment induces ER-stress response. KBM5 and KBM5R cells were treated with 0.75 μM NiPT for the indicated duration, then cell lysates were immunoblotted to assess the changes of p-eIF2α and CHOP